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Aminomethylphosphonate (NMePo) and 2-aminoethylphosphonate (NEthPo) were evaluated as alternative pH indicators in the isolated perfused rat liver using ³¹P-nuclear magnetic resonance (NMR). NMePo did not distribute within cells and remained in the extracellular space. It exhibited pH titration with a low pKa value (5.35). This behaviour makes NMePo useful for extracellular volume or acidic pH determination. In contrast, NEthPo accumulated within cells without altering liver energetic steady state, evaluated from nucleosides triphosphates resonances, even for prolonged (100 min) experiments. Withdrawal of NEthPo from perfusate revealed a residual resonance corresponding to the internalized amount of this phosphonate. This fraction was almost stable vs time and allowed determination of spin-lattice relaxation time constant T1 within the liver (2.2 ± 0.3 s; n = 6). Comparison of the titration curves for NEthPo and inorganic phosphate revealed that the accuracy of pH determination within physiologic or acidic range in both cases was comparable. Finally, when extracellular pH was decreased, the NEthPo resonance frequency was found to undergo the same chemical shift variations as observed for cytosolic Pi signal, which was in good agreement with a cytosolic accumulation of this phosphonate. Therefore, NEthPo could be considered as an interesting cytosolic pH probe suitable for ³¹P-NMR measurements, especially when experimental conditions prevent reliable observation of cytosolic Pi resonance. Copyright © 2000 John Wiley & Sons, Ltd. Abbreviations used: KH Krebs–Henseleit buffer MePoMe2 dimethyl methylphosphonate NEthPo 2-aminoethylphosphonate NMePo aminomethylphosphonate NProPo 3-aminopropylphosphonate NTP nucleoside triphosphates Pi inorganic phosphate pHin cytosolic pH pHout effluent pH

Aminomethylphosphonate (NMePo) and 2‐aminoethylphosphonate (NEthPo) were evaluated as alternative pH indicators in the isolated perfused rat liver using 31P‐nuclear magnetic resonance (NMR). NMePo did not distribute within cells and remained in the extracellular space. It exhibited pH titration with a low pKavalue (5.35). This behaviour makes NMePo useful for extracellular volume or acidic pH determination. In contrast, NEthPo accumulated within cells without altering liver energetic steady state, evaluated from nucleosides triphosphates resonances, even for prolonged (100 min) experiments. Withdrawal of NEthPo from perfusate revealed a residual resonance corresponding to the internalized amount of this phosphonate. This fraction was almost stable vs time and allowed determination of spin‐lattice relaxation time constant T1within the liver (2.2 ± 0.3 s; n= 6). Comparison of the titration curves for NEthPo and inorganic phosphate revealed that the accuracy of pH determination within physiologic or acidic range in both cases was comparable. Finally, when extracellular pH was decreased, the NEthPo resonance frequency was found to undergo the same chemical shift variations as observed for cytosolic Pisignal, which was in good agreement with a cytosolic accumulation of this phosphonate. Therefore, NEthPo could be considered as an interesting cytosolic pH probe suitable for 31P‐NMR measurements, especially when experimental conditions prevent reliable observation of cytosolic Pi resonance. Copyright © 2000 John Wiley & Sons, Ltd.